Project Summary/Abstract One of the most exciting biotechnological breakthroughs in the past decade was the discovery of the genome editing toolbox CRISPR-Cas9 system. Although the current applications of CRISPR-Cas9 focus on basic biological research, this technology holds promise to revolutionize the drug discovery process for many inherited disorders including disorders in hematopoietic system. However, off-target mismatches that lead to unintended genome changes become one of major obstacles faced by the potential clinical applications of CRISPR-Cas9. Modulations of CRISPR-Cas9 have been reported to enhance its specificity. But these approaches are only partially effective. New approaches to increase CRISPR-Cas9 specificity are greatly needed for precise genome editing. Understanding of molecular mechanisms of CRSIPR-Cas9 will greatly facilitate the development of these new approaches, but lack of structural information of how Cas9 interact with DNA in its active state makes the mechanism study of CRISPR-Cas9 still in its infancy. Our preliminary study has captured the active state conformation of Cas9 and enabled the identification of key residues involved in Cas9 specificity with powerful computational tools. In this application, we hypothesize that Cas9 activation is allosterically regulated and its specificity can be allosterically modulated. Our goal is to elucidate the mechanisms of CRISPR-Cas9 and develop new approaches to enhance its specificity using a combination of computational and experimental methods. In Aim 1, we propose to elucidate the allosteric mechanism for CRISPR-Cas9 activation. We will characterize the active states and identify key residues at the active sites and allosteric sites that regulating Cas9 activation, using a combination of computational and experimental methods. Aim 2 focuses on specificity. The mechanisms of specificity will be explored and new Cas9 variants will be designed and tested for their activity and specificity. Together, our efforts will provide a foundation to understand the molecular mechanisms of CRISPR- Cas9 specificity. The designed new Cas9 variants will provide new approaches to improve CRISPR- Cas9 technology and advance precise genome editing.